This invention relates to compositions, apparatus and methods for detecting one or more nucleic acid targets present in a sample. The detection of specific nucleic acids is an important tool for diagnostic medicine and molecular biology research.
Gene probe assays currently play roles in identifying infectious organisms such as bacteria and viruses, in probing the expression of normal and mutant genes and identifying genes associated with disease or injury, such as oncogenes, in typing tissue for compatibility preceding tissue transplantation, in matching tissue or blood samples for forensic medicine, for responding to emergency response situations like a nuclear incident or pandemic flu outbreak, in determining disease prognosis or causation, and for exploring homology among genes from different species.
Ideally, a gene probe assay should be sensitive, specific and easily automatable (for a review, see Nickerson, Current Opinion in Biotechnology (1993) 4:48-51.) The requirement for sensitivity (i.e. low detection limits) has been greatly alleviated by the development of the polymerase chain reaction (PCR) and other amplification technologies which allow researchers to exponentially amplify a specific nucleic acid sequence before analysis (for a review, see Abramson et al., Current Opinion in Biotechnology, (1993) 4:41-47). For example, multiplex PCR amplification of SNP loci with subsequent hybridization to oligonucleotide arrays has been shown to be an accurate and reliable method of simultaneously genotyping hundreds of SNPs (see Wang et al., Science, (1998) 280:1077; see also Schafer et al., Nature Biotechnology, (1989)16:33-39).
Specificity also remains a problem in many currently available gene probe assays. The extent of molecular complementarity between probe and target defines the specificity of the interaction. Variations in composition and concentrations of probes, targets and salts in the hybridization reaction as well as the reaction temperature, and length of the probe may all alter the specificity of the probe/target interaction.
It may be possible under some circumstances to distinguish targets with perfect complementarity from targets with mismatches, although this is generally very difficult using traditional technology, since small variations in the reaction conditions will alter the hybridization. Newer techniques with the necessary specificity for mismatch detection include probe digestion assays in which mismatches create sites for probe cleavage, and DNA ligation assays where single point mismatches prevent ligation.
A variety of enzymatic and non-enzymatic methods are available for detecting sequence variations. Examples of enzyme based methods include Invader™, oligonucleotide ligation assay (OLA) single base extension methods, allelic PCR, and competitive probe analysis (e.g. competitive sequencing by hybridization). Enzymatic DNA ligation reactions are well known in the art (Landegren, Bioessays (1993) 15(11):761-5; Pritchard et al., Nucleic Acids Res. (1997) 25(17):3403-7; Wu et al., Genomics, (1989) 4(4):560-9) and have been used extensively in SNP detection, enzymatic amplification reactions and DNA repair.
A number of non-enzymatic or template mediated chemical ligation methods have been developed that can be used to detect sequence variations. These include chemical ligation methods that utilize coupling reagents, such as N-cyanoimidazole, cyanogen bromide, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. See Metelev, V. G., et al., Nucleosides & Nucleotides (1999) 18:2711; Luebke, K. J., and Dervan, P. B. J. Am. Chem. Soc. (1989) 111:8733; and Shabarova, Z. A., et al., Nucleic Acids Research (1991)19:4247, each of which is incorporated herein by reference in its entirety.
Kool (U.S. Pat. No. 7,033,753), which is incorporated herein by reference in its entirety describes the use of chemical ligation and fluorescence resonance energy transfer (FRET) to detect genetic polymorphisms. The readout in this process is based on the solution phase change in fluorescent intensity.
Terbrueggen (U.S. Patent application 60/746,897) which is incorporated herein by reference in its entirety describes the use of chemical ligation methods, compositions and reagents for the detection of nucleic acids via microarray detection.
Other chemical ligation methods react a 5′-tosylate or 5′-iodo group with a 3′-phosphorothioate group, resulting in a DNA structure with a sulfur replacing one of the bridging phosphodiester oxygen atoms. See Gryanov, S. M., and Letsinger, R. L., Nucleic Acids Research (1993) 21:1403; Xu, Y. and Kool, E. T. Tetrahedron Letters (1997) 38:5595; and Xu, Y. and Kool, E. T., Nucleic Acids Research (1999) 27:875, each of which is herein incorporated by reference in its entirety.
Some of the advantages of using non-enzymatic approaches for nucleic acid target detection include lower sensitivity to non-natural DNA analog structures, ability to use RNA target sequences, lower cost and greater robustness under varied conditions. Letsinger et al (U.S. Pat. No. 5,780,613, herein incorporated by reference in its entirety) have previously described an irreversible, nonenzymatic, covalent autoligation of adjacent, template-bound oligonucleotides wherein one oligonucleotide has a 5′ displaceable group and the other oligonucleotide has a 3′ thiophosphoryl group.
PCT applications WO 95/15971, PCT/US96/09769, PCT/US97/09739, PCT US99/01705, WO96/40712 and WO98/20162, all of which are expressly incorporated herein by reference in their entirety, describe novel compositions comprising nucleic acids containing electron transfer moieties, including electrodes, which allow for novel detection methods of nucleic acid hybridization.
One technology that has gained increased prominence involves the use of DNA arrays (Marshall et al., Nat Biotechnol. (1998) 16(1):27-31), especially for applications involving simultaneous measurement of numerous nucleic acid targets. DNA arrays are most often used for gene expression monitoring where the relative concentration of 1 to 100,000 nucleic acids targets (mRNA) is measured simultaneously. DNA arrays are small devices in which nucleic acid anchor probes are attached to a surface in a pattern that is distinct and known at the time of manufacture (Marshall et al., Nat Biotechnol. (1998) 16(1):27-31) or can be accurately deciphered at a later time such as is the case for bead arrays (Steemers et al., Nat Biotechnol. (2000) 18(1):91-4; and Yang et al., Genome Res. (2001) 11(11):1888-98.). After a series of upstream processing steps, the sample of interest is brought into contact with the DNA array, the nucleic acid targets in the sample hybridize to anchor oligonucleotides on the surface, and the identity and often concentration of the target nucleic acids in the sample are determined.
Many of the nucleic acid detection methods in current use have characteristics and/or limitations that hinder their broad applicability. For example, in the case of DNA microarrays, prior to bringing a sample into contact with the microarray, there are usually a series of processing steps that must be performed on the sample. While these steps vary depending upon the manufacturer of the array and/or the technology that is used to read the array (fluorescence, electrochemistry, chemiluminescence, magnetoresistance, cantilever deflection, surface plasmon resonance), these processing steps usually fall into some general categories: Nucleic acid isolation and purification, enzymatic amplification, detectable label incorporation, and clean up post-amplification. Other common steps are sample concentration, amplified target fragmentation so as to reduce the average size of the nucleic acid target, and exonuclease digestion to convert PCR amplified targets to a single stranded species.
The requirement of many upstream processing steps prior to contacting the DNA array with the sample can significantly increase the time and cost of detecting a nucleic acid target(s) by these methods. It can also have significant implications on the quality of the data obtained. For instance, some amplification procedures are very sensitive to target degradation and perform poorly if the input nucleic acid material is not well preserved (Foss et al., Diagn Mol Pathol. (1994) 3(3):148-55). Technologies that can eliminate or reduce the number and/or complexity of the upstream processing steps could significantly reduce the cost and improve the quality of results obtained from a DNA array test. One method for reducing upstream processing steps involves using ligation reactions to increase signal strength and improve specificity.
There remains a need for methods and compositions for efficient and specific nucleic acid detection. Accordingly, the present invention provides methods and compositions for non-enzymatic chemical ligation reactions which provides very rapid target detection and greatly simplified processes of detecting and measuring nucleic acid targets.